Rett syndrome (RTT) is a severe neurodevelopmental disorder that is caused by mutations in the methyl-DNA- binding protein MeCP2 and represents one of the most common causes of intellectual disability in females. RTT symptoms include a progressive loss of speech and social engagement, seizures, stereotyped hand movements, and motor-system disabilities. The monogenic etiology of RTT has enabled the development of MeCP2-mutant mouse models that display synaptic and behavioral phenotypes that reasonably accurately model RTT symptoms. Importantly, re-expression of MeCP2 in adult MeCP2-null mice reverses the synaptic and behavioral phenotypes, suggesting the possibility of ameliorating the cognitive and behavioral difficulties of girls with RTT. However, the incomplete understanding of MeCP2 molecular function limits therapeutic development. The proposed study will use new mouse models and approaches to characterize the immediate consequences of MeCP2 loss and to investigate key neuronal activity-dependent components of MeCP2 function. Aim 1: Of the many molecular consequences associated with constitutive loss of MeCP2, researchers do not yet know which are direct results of MeCP2 loss and which are secondary consequences of long-term neurological impairment. Dr. Boxer will use a new mouse line that enables rapid and specific degradation of the MeCP2 protein to characterize the molecular, cellular, and behavioral consequences of acute loss of MeCP2 in the brain. These experiments will reveal the relative order of phenotype appearance, which will enable the identification of the direct molecular effects of MeCP2 loss and provide insight into the synaptic and behavioral steps of disease progression. Aim 2: MeCP2 is rapidly phosphorylated at multiple sites in response to neuronal activity, and MeCP2-null neurons display defects in experience-dependent synaptic refinement. However, the synaptic refinement phenotype has not been directly or immediately connected to activity-dependent MeCP2 phosphorylation. Dr. Boxer will use a recently developed mouse line with alanine mutations in all activity- dependent phosphorylation sites to investigate the role of activity-dependent MeCP2 phosphorylation in regulation of gene expression and synaptic refinement. Overall, these experiments will provide temporally and mechanistically precise answers to key outstanding questions in MeCP2 research, potentially enabling the development of targeted therapeutics for RTT. The proposed research will provide training for Dr. Boxer in electrophysiology and bioinformatics, and more broadly in neurobiology and career development, in the laboratory of her mentor, Dr. Michael Greenberg, and with guidance from her Advisory Committee at Harvard Medical School. Dr. Boxer intends to become an academic professor studying the molecular basis of neurodevelopmental disorders.